This Small Business Innovation Research (SBIR) Phase I project aims to develop a microfluidic-based nitric oxide (NO) sensor as an early sepsis risk assessment device. Sepsis causes significant strain on the U.S. healthcare system, consuming over $17 billion annually due to extended hospital stays, and significant morbidity and mortality. Rapid diagnosis and intervention are critical to improve patient outcomes. Unfortunately, current methods of diagnosing sepsis rely on the detection of symptoms (e.g., fever and irregular heart rate) that only become evident after the infection has progressed to dangerous levels (advanced sepsis). The goals of this project are to: 1) manufacture prototype miniaturized microfluidic sensors for the measurement of NO levels in drawn blood; and, 2) determine the potential clinical utility of NO determination in preclinical, monomicrobial models of pulmonary sepsis. Using indirect analysis methods, literature studies have found significant elevations (>10x basal) in blood nitric oxide due to sepsis. However, the lack of a rapid and direct sensor for NO has frustrated the translation of these findings to the clinic. Initial studie in a porcine model of polymicrobial sepsis have shown that this microfluidic NO sensor can represents a new paradigm for diagnosing early sepsis and saving lives. This finding is supported by in vitro studies showing that macrophages release NO in response to bacteria, with increasing magnitude according to bacteria load. Through the proposed studies we will develop a novel, miniaturized NO sensing platform to enable the rapid measurement of this biomarker in small aliquots of blood. After meeting specific analytical performance requirements for this device (sensitivity and selectivity for NO, linear response range, reproducibility, etc.),we will determine the predictive value of NO measurement in established preclinical models of pulmonary sepsis. Most in vivo studies of NO in sepsis have been performed in polymicrobial models such as cecal ligation and puncture (CLP). While this model faithfully recapitulates ruptured appendicitis, the microbiological milieu is more complex than most hospital-acquired infections (i.e., most catheter-related infections and cases of hospital-acquired pneumonia have a single etiologic agent). Both clinical pneumonia and the preclinical, murine models of pneumonia share common immunoinflammatory elements and, in contrast to CLP, are highly reproducible and titratable. These models will allow testing of several clinically significant parameters, including: 1) differentiating the blood nitric oxide response in Gram- positive and Gram-negative infections, 2) determining whether the in vivo nitric oxide response is proportional to microbial burden, and 3) screening commonly used therapeutic modalities (for example, pain management and antibiotics) to assess their affects on the blood nitric oxide response to infection. These studies will establish key parameters for the evaluation of the blood nitric oxide sensor in clinical subjects.